Identification and characterization of an ecto-pyrophosphatase activity in intact epimastigotes of Trypanosoma rangeli

• Published in: PloS one 2014-09, Vol.9 (9), p.e106852-e106852
• Main Authors: Fonseca-de-Souza, André Luiz, Freitas-Mesquita, Anita Leocadio, Vieira, Lisvane Paes, Majerowicz, David, Daflon-Yunes, Nathalia, Soares-de-Medeiros, Lia Carolina Almeida, Miranda, Kildare, Gondim, Katia Calp, Meyer-Fernandes, José Roberto
• Format: Article
• Language: English
• Subjects: Biology and Life Sciences; Calcium chloride; Cell culture; Cell Proliferation; Cytometry; Diphosphates - metabolism; Energy; Epimastigotes; Flow cytometry; Fluoride; Fluorides; Homeostasis; Hydrogen; Hydrolysis; Immunofluorescence; Inorganic Pyrophosphatase - metabolism; Kinases; Life Cycle Stages; Magnesium chloride; Membranes; Metabolism; Microscopy; Molecular chains; Parasites; Phosphatase; Phosphates; Proteins; Protozoa; Pyrophosphatase; Sodium; Sodium fluoride; Trends; Trypanosoma brucei; Trypanosoma rangeli; Trypanosoma rangeli - cytology; Trypanosoma rangeli - enzymology; Trypanosoma rangeli - growth & development
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0106852

In this study, we performed the molecular and biochemical characterization of an ecto-enzyme present in Trypanosoma rangeli that is involved with the hydrolysis of extracellular inorganic pyrophosphate. PCR analysis identified a putative proton-pyrophosphatase (H(+)-PPase) in the epimastigote forms of T. rangeli. This protein was recognized with Western blot and flow cytometry analysis using an antibody against the H(+)-PPase of Arabidopsis thaliana. Immunofluorescence microscopy confirmed that this protein is located in the plasma membrane of T. rangeli. Biochemical assays revealed that the optimum pH for the ecto-PPase activity was 7.5, as previously demonstrated for other organisms. Sodium fluoride (NaF) and aminomethylenediphosphonate (AMDP) were able to inhibit approximately 75% and 90% of the ecto-PPase activity, respectively. This ecto-PPase activity was stimulated in a dose-dependent manner by MgCl2. In the presence of MgCl2, this activity was inhibited by millimolar concentrations of CaCl2. The ecto-PPase activity of T. rangeli decreased with increasing cell proliferation in vitro, thereby suggesting a role for this enzyme in the acquisition of inorganic phosphate (Pi). Moreover, this activity was modulated by the extracellular concentration of Pi and increased approximately two-fold when the cells were maintained in culture medium depleted of Pi. All of these results confirmed the occurrence of an ecto-PPase located in the plasma membrane of T. rangeli that possibly plays an important role in phosphate metabolism of this protozoan.